The goal of Phase I of this SBIR application is to establish the feasibility of using a newly invented gene silencing technology called U1 Adaptors that uses Adaptor oligonucleotides to silence the human BCL2 gene, a key player in tumor progression. The long term goal is to use anti-BCL2 Adaptors to treat patients with various types of cancers, with a first target being lung cancer. Currently two types of oligonucleotide-based gene silencing methods are being pursued as a therapeutic method. Traditional approaches use an antisense oligonucleotide (ASO) designed to base pair with its complementary target mRNA leading to either degradation or impaired function of the mRNA. Current excitement has focused on RNAi that uses a distinct mechanism where siRNA oligonucleotides trigger an endogenous pre-existing gene suppression pathway. In spite of the general success of siRNA, some genes are only modestly downregulated (<2-fold) and others are refractory, and certain off-target effects can arise leading to unexpected consequences, underscoring the need for additional methods. The founders of SilaGene have invented a new method that uses U1 Adaptor oligonucleotides to anneal to specific sequence regions within the 3'terminal exon to inhibit pre-mRNA processing (ie. polyA tail addition). U1 Adaptors are superior to siRNAs and ASOs in terms of bioavailability (due to increased stability) and delivery (due to accepting a higher degree of targeting groups). An additional attractive feature is U1 Adaptors give enhanced silencing when used in combination with each other or with siRNAs. The specific aims of Phase I are to develop an "Adaptor/nanocarrier" system that combines a nanocarrier delivery system with U1 Adaptors with the goal being to identify the best, most active anti-BCL2 Adaptors to silence the human BCL2 gene in cell culture. This will be followed by a pilot experiment to test the Adaptor/nanocarrier system in a "xenograft mouse" system involving growing a human tumor in mice. Phase I will also assess the genome-wide specificity of the anti-BCL2 Adaptor/nanocarrier system in cell culture using microarray analysis. In Phase II, the best Adaptors from Phase I will be extensively and fully tested in the same xenograft mouse system to assess their: 1) potency in silencing BCL2, 2) efficacy in suppressing tumor growth, and 3) cytotoxicity. PUBLIC HEALTH RELEVANCE: The commercialization of the U1 Adaptor technology will be a significant addition to the scientific research community's "gene silencing toolkit". U1 Adaptor oligonucleotides are superior to siRNAs because they are much more stable and deliverable to the target tissue or organ. This is due to the fact that U1 Adaptors accept a far higher degree of targeting groups and chemical modifications as compared to siRNAs. Because U1 Adaptors exploit a distinctly different mechanism compared to more common gene silencing approaches, it has the potential of enhancing these traditional technologies when used in combination with them via synergistic effects. The achievement of the long term goal of this SBIR proposal, namely to silence BCL2, will give a therapeutically beneficial suppression of tumor growth in cancer patients.